Unit: Mouse Molecular Genetics - URA CNRS 2578
Director: Philip AVNER
Research in the Unit is centred around three topics :
1) Epigenetics and the Inactivation of the mouse X chromosome
2) Multifactorial Genetics and the Genetic analysis of type 1 diabetes in the mouse.
3) Stem Cells and the role of the X-linked gene Nap1l2 in the control of neuronal stem cell proliferation
1) Epigenetics and the Inactivation of the mouse X chromosome
One of the major research interests of the Mouse Molecular Genetics Unit is X chromosome inactivation. This is a complex biological process which depends on the presence of the X-inactivation centre (Xic) and which results in the inactivation of one of the two X chromosomes present in the female cell. The process involves both the sensing and counting of the number of X chromosomes in the cell in relation to the autosomal complement and a choice process concerning the X to inactivate. The Xic increasingly appears as a complex control region with multiple elements feeding into the regulation and interactions with the Xist (X-inactive specific transcript) gene which codes for a large non-coding RNA which plays a critical role in the initiation of the inactivation process.
A major part of the work in the laboratory is concerned with the functional identification and analysis of the various components of the Xic involved in the counting and choice mechanisms. Our strategy exploits the creation of novel mutations by targeted mutagenesis using the cre-lox system. We have been able to establish by this approach the key role played by the Tsix antisense in the processus of counting though its mediation of Xist activity and regulation. Analysis of sequence variation and conservation of the Xic are being explored in collaboration with the Genoscope (Evry) and Laurent Duret (Lyon University) in a project involving the in-depth sequencing and annotation of the Xic of seven mammalian species. It is hoped that the approach will also lead to indications as to the evolutionary origins of the non coding Xist RNA.
A better understanding of some of the processes involved in the initiation of X-inactivation is likely to depend on combining such genetic and cell biology approaches with a detailed analysis of chromatin structure. Several different approaches to such an analysis including ChIP (Chromatin Immunoprecipitation) have been established in the laboratory. An example of such an approach is a recent study involving Chromatin immunoprecipitation (ChiP) analysis of ES strains carrying genetically engineeered modifications of the 3' part of the Xic which has established that the Xist antisense transcript Tsix affects Xist transcription indirectly, rather than directly, through its effects on chromatin structure and especially chromatin structure around the Xist promoter. ChIP studies have also proved of great importance in characterising the role played by H3 histone modifications in the earliest stages of X-inactivation and have allowed us to define a region 5' to Xist which may act as a nucleation centre allowing the inactivation to spread from the Xic over the whole X chromosome. The elements defined by these studies are being characterised by genetic modification and analysis ex vivo and in vivo of the resulting material. Promising results have been obtained.
Our analysis of trans-acting genetic factors involved in the X-inactivation process has been recentred around facteurs involved in RNA metabolism. Our functional analysis approach to these candidates which involves RNAi vectors allowing stable knockdown of expression and observation of altered X-inactivation parameters, has allowed yet a further level of Xist regulation to be defined.
Genome Research and Mouse Disease Models
2) The Genetic analysis of type 1 diabetes
Another of the Unit's interest concerns the study of mouse phenotypes under multifactorial and polygenic control. We have taken as our prototype, type 1 diabetes or insulin- dependent diabetes for which the NOD mouse represents an interesting model. Our studies are aimed at defining the genetic factors (Idd) implicated in this pathology which is known to depend on a complex interaction between environmental and genetic factors. Our studies are concentrated on the characterisation of Idd loci controlling diabetes susceptibility/resistance locating to the distal part of mouse chromosome 6.
The continued refining of the candidate regions for these loci by establishment of congenic mouse strains has allowed candidate gene approaches to be undertaken to defining and characterising the genes responsible for these traits and in particular for Idd6. Varied approaches including transcriptional profiling have exploited these results and this has allowed five candidate genes for the Idd6 locus to be defined. The best defined gene implicated in the diabetes susceptibility/resistance turns out to be important in the control of diurnal rhythyms, opening up a whole new biological network for its role in the etiology of diabetes in mouse, and eventually in man. This gene and the other candidates is currently under functional analysis using RNAi lentivirus based approaches.
3) Stem Cells and the role of the X-linked gene Nap1l2
A second disease subject to both environmental and complex genetic control is spina bifida. In the mouse spina bifida can be induced by mutation in the X-linked Nap1l2 gene. Mutations in this gene are associated with embryonic lethality, spina bifida and exencephaly linked to a massive overproliferation of neuronal cells. We have now been able to obtain conditional mutations in the Nap1l2 gene and these are being exploited to explore its role in the adult mouse. The recent establishment of a highly efficient system for differentiating ES cells into neural stem cells and cells of the neuronal lineage has opened up interesting perspective for understanding the role and mechanism of action of this gene, its interactions and its cellular partners. Our studies have suggested that Nap1l2 controls gene activity in the neuronal lineage via an action on histone modification of its target genes.
Keywords: Epigenetics, X chromosome inactivation, Chromatin, Genetics, Genomics, Type 1 Diabetes, Mouse, QTL, Multigenic inheritance, Stem cells